Parkinson's disease is a widespread condition caused by the loss of midbrain dopaminergic neurons. Human embryonic stem cells (hESC) can be directed to differentiate into midbrain dopaminergic neurons in vitro, and are a potentially unlimited source of replacement cells. However, there is a growing demand for improved culture methods leading to greater yield, and with sufficient safety to allow for clinical trials. Human ESC are difficult to grow and differentiate to dopaminergic cells, and the lengthy protocols require exposure of the cells to potential sources of animal pathogens and antigens, such as animal serum and mouse feeder cells. The fibroblasts used as feeder cells are also an unwanted contaminant in transcriptome analyses of hESC pluripotency. Therefore, by using standard molecular biology methods we will create and test plasmid vectors which can express a stable marker on the surface of feeder cells. These markers will allow the feeders to be selectively depleted from hESC cultures. Specific Aims: 1. To test and modify vector systems for the stable and efficient expression of selective markers in mouse and human fibroblasts. 2. To develop immortal MEF and HFF lines that can be easily discriminated and removed from human and mouse ESC via magnetic separation and fluorescence-activated cell sorting (FACS). 3. To expand modified and tagged feeder cell lines those are validated for the ability to support the undifferentiated state of hESC. Human Embryonic Stem Cells: This work will use the NIH-approved hESC line BG01. Relevance: Parkinson's disease afflicts about 1 million Americans. This disorder is caused by the loss of a specific type of neuron in the brain which controls voluntary movements. Human embryonic stem cells (hESC) are capable of becoming any cell type of the body including these dopaminergic neurons, but are demanding for investigators to grow. This project will produce a cell which supports the growth of hESC in culture and will improve the generation of dopaminergic neurons, with safety features required for future clinical research. [unreadable] [unreadable]